The photomorphogenic repressors BBX28 and BBX29 integrate light and brassinosteroid signaling to inhibit seedling development in Arabidopsis.

B-box containing proteins (BBXs) integrate light and various hormonal signals to regulate plant growth and development. Here, we demonstrate that the photomorphogenic repressors BBX28 and BBX29 positively regulate brassinosteroid (BR) signaling in Arabidopsis thaliana seedlings. Treatment with the BR brassinolide stabilized BBX28 and BBX29, which partially depended on BR INSENSITIVE1 (BRI1) and BIN2. bbx28 bbx29 seedlings exhibited larger cotyledon aperture than the wild-type when treated with brassinazole in the dark, which partially suppressed the closed cotyledons of brassinazole resistant 1-1D (bzr1-1D). Consistently, overexpressing BBX28 and BBX29 partially rescued the short hypocotyls of bri1-5 and bin2-1 in both the dark and light, while the loss-of-function of BBX28 and BBX29 partially suppressed the long hypocotyls of bzr1-1D in the light. BBX28 and BBX29 physically interacted with BR-ENHANCED EXPRESSION1 (BEE1), BEE2, and BEE3 and enhanced their binding to and activation of their target genes. Moreover, BBX28 and BBX29 as well as BEE1, BEE2, and BEE3 increased BZR1 accumulation to promote the BR signaling pathway. Therefore, both BBX28 and BBX29 interact with BEE1, BEE2, and BEE3 to orchestrate light and BR signaling by facilitating the transcriptional activity of BEE target genes. Our study provides insights into the pivotal roles of BBX28 and BBX29 as signal integrators in ensuring normal seedling development.

Jasmonates regulate apical hook development by repressing brassinosteroid biosynthesis and signaling.

An apical hook is a special structure formed during skotomorphogenesis in dicotyledonous plant species. It is critical for protecting the shoot apical meristem from mechanical damage during seed germination and hypocotyl elongation in soil. Brassinosteroid (BR) and jasmonate (JA) phytohormones antagonistically regulate apical hook formation. However, the interrelationship between BRs and JAs in this process has not been well elucidated. Here, we reveal that JAs repress BRs to regulate apical hook development in Arabidopsis (Arabidopsis thaliana). Exogenous application of methyl jasmonate (MeJA) repressed the expression of the rate-limiting BR biosynthetic gene DWARF4 (DWF4) in a process relying on 3 key JA-dependent transcription factors, MYC2, MYC3, and MYC4. We demonstrated that MYC2 interacts with the critical BR-activated transcription factor BRASSINAZOLE RESISTANT 1 (BZR1), disrupting the association of BZR1 with its partner transcription factors, such as those of the PHYTOCHROME INTERACTING FACTOR (PIF) family and downregulating the expression of their target genes, such as WAVY ROOT GROWTH 2 (WAG2), encoding a protein kinase essential for apical hook development. Our results indicate that JAs not only repress the expression of BR biosynthetic gene DWF4 but, more importantly, attenuate BR signaling by inhibiting the transcriptional activation of BZR1 by MYC2 during apical hook development.

Knock out of amino acid transporter gene OsLHT1 accelerates leaf senescence and enhances resistance to rice blast fungus.

Plant amino acid transporters regulate not only long-distance transport and reallocation of nitrogen (N) from source to sink organs, but also the amount of amino acids in leaves hijacked by invading pathogens. However, the function of amino acid transporters in plant defense responses to pathogen infection remains unknown. In this study, we found that the rice amino acid transporter gene OsLHT1 was expressed in leaves and up-regulated by maturation, N starvation, and inoculation of the blast fungus Magnaporthe oryzae. Knock out of OsLHT1 resulted in development stage- and N supply-dependent premature senescence of leaves at the vegetative growth stage. In comparison with the wild type, Oslht1 mutant lines showed sustained rusty red spots on fully mature leaf blades irrespective of N supply levels. Notably, no relationship between the severity of leaf rusty red spots and concentration of total N or amino acids was found in Oslht1 mutants at different developmental stages. Disruption of OsLHT1 altered transport and metabolism of amino acids and biosynthesis of flavones and flavonoids, enhanced expression of jasmonic acid- and salicylic acid-related defense genes, production of jasmonic acid and salicylic acid, and accumulation of reactive oxygen species. OsLHT1 inactivation dramatically prevented the leaf invasion by M. oryzae, a hemi-biotrophic ascomycete fungus. Overall, these results establish a link connecting the activity of an amino acid transporter with leaf metabolism and defense against rice blast fungus.

Creation of fragrant sorghum by CRISPR/Cas9.

Sorghum, the fifth largest cereal crop, has high value as a staple food and raw material for liquor and vinegar brewing. Due to its high biomass and quality, it is also used as the second most planted silage resource. No fragrant sorghums are currently on the market. Through CRISPR/Cas9-mediated knockout of SbBADH2, we obtained sorghum lines with extraordinary aromatic smell in both seeds and leaves. Animal feeding experiments showed that fragrant sorghum leaves were attractable. We believe this advantage will produce great value in the sorghum market for both grain and whole biomass forage.

Structural insights into a novel nonheme iron-dependent oxygenase in selenoneine biosynthesis.

Selenoneine (SEN) is a natural histidine derivative with radical-scavenging activity and shows higher antioxidant potential than its sulfur-containing isolog ergothioneine (EGT). Recently, the SEN biosynthetic pathway in Variovorax paradoxus was reported. Resembling EGT biosynthesis, the committed step of SEN synthesis is catalyzed by a nonheme Fe-dependent oxygenase termed SenA. This enzyme catalyzes oxidative carbon­selenium (C-Se) bond formation to conjugate N-α-trimethyl histidine (TMH) and selenosugar to yield selenoxide; the process parallels the EGT biosynthetic route, in which sulfoxide synthases known as EgtB members catalyze the conjugation of TMH and cysteine or γ-glutamylcysteine to afford sulfoxides. Here, we report the crystal structures of SenA and its complex with TMH and thioglucose (SGlc), an analog of selenoglucose (SeGlc) at high resolution. The overall structure of SenA adopts the archetypical fold of EgtB, which comprises a DinB-like domain and an FGE-like domain. While the TMH-binding site is highly conserved to that of EgtB, a various substrate-enzyme interaction network in the selenosugar-binding site of SenA features a number of water-mediated hydrogen bonds. The obtained structural information is beneficial for understanding the mechanism of SenA-mediated C-Se bond formation.

Remodeling the polymer-binding cavity to improve the efficacy of PBAT-degrading enzyme.

Poly(butylene adipate-co-terephthalate) (PBAT) is among the most widely applied synthetic polyesters that are utilized in the packaging and agricultural industries, but the accumulation of PBAT wastes has posed a great burden to ecosystems. Using renewable enzymes to decompose PBAT is an eco-friendly solution to tackle this problem. Recently, we demonstrated that cutinase is the most effective PBAT-degrading enzyme and that an engineered cutinase termed TfCut-DM could completely decompose PBAT film to terephthalate (TPA). Here, we report crystal structures of a variant of leaf compost cutinase in complex with soluble fragments of PBAT, including BTa and TaBTa. In the TaBTa complex, one TPA moiety was located at a polymer-binding site distal to the catalytic center that has never been experimentally validated. Intriguingly, the composition of the distal TPA-binding site shows higher diversity relative to the one proximal to the catalytic center in various cutinases. We thus modified the distal TPA-binding site of TfCut-DM and obtained variants that exhibit higher activity. Notably, the time needed to completely degrade the PBAT film to TPA was shortened to within 24 h by TfCut-DM Q132Y (5813 mol per mol protein). Taken together, the structural information regarding the substrate-binding behavior of PBAT-degrading enzymes could be useful guidance for direct enzyme engineering.

Tomato CYP94C1 inactivates bioactive JA-Ile to attenuate jasmonate-mediated defense during fruit ripening.

As the master regulators of the ET signaling pathway, EIL transcription factors directly activate the expression of CYP94C1 to inactivate bioactive JA-Ile, thereby attenuating JA-mediated defense during fruit ripening. Knockout of CYP94C1 improves tomato fruit resistance to necrotrophs without compromising fruit quality.

SnoRNP is essential for thermospermine-mediated development in Arabidopsis thaliana.

Polyamines have been discovered for hundreds of years and once considered as a class of phytohormones. Polyamines play critical roles in a range of developmental processes. However, the molecular mechanisms of polyamine signaling pathways remain poorly understood. Here, we measured the contents of main types of polyamines, and found that endogenous level of thermospermine (T-Spm) in Arabidopsis thaliana is comparable to those of classic phytohormones and is significantly lower than those of putrescine (Put), spermidine (Spd), and spermine (Spm). We further found a nodule-like structure around the junction area connecting the shoot and root of the T-Spm biosynthetic mutant acl5 and obtained more than 50 suppressors of acl5nodule structure (san) through suppressor screening. An in-depth study of two san suppressors revealed that NAP57 and NOP56, core components of box H/ACA and C/D snoRNPs, were essential for T-Spm-mediated nodule-like structure formation and plant height. Furthermore, analyses of rRNA modifications showed that the overall levels of pseudouridylation and 2'-O-methylation were compromised in san1 and san2 respectively. Taken together, these results establish a strong genetic relationship between rRNA modification and T-Spm-mediated growth and development, which was previously undiscovered in all organisms.

Brassinosteroid coordinates cell layer interactions in plants via cell wall and tissue mechanics.

Growth coordination between cell layers is essential for development of most multicellular organisms. Coordination may be mediated by molecular signaling and/or mechanical connectivity between cells, but how genes modify mechanical interactions between layers is unknown. Here we show that genes driving brassinosteroid synthesis promote growth of internal tissue, at least in part, by reducing mechanical epidermal constraint. We identified a brassinosteroid-deficient dwarf mutant in the aquatic plant Utricularia gibba with twisted internal tissue, likely caused by mechanical constraint from a slow-growing epidermis. We tested this hypothesis by showing that a brassinosteroid mutant in Arabidopsis enhances epidermal crack formation, indicative of increased tissue stress. We propose that by remodeling cell walls, brassinosteroids reduce epidermal constraint, showing how genes can control growth coordination between layers by means of mechanics.

A Tailored High-Efficiency Sample Pretreatment Method for Simultaneous Quantification of 10 Classes of Known Endogenous Phytohormones.

One of the hottest topics in plant hormone biology is the crosstalk mechanisms, whereby multiple classes of phytohormones interplay with each other through signaling networks. To better understand the roles of hormonal crosstalks in their complex regulatory networks, it is of high significance to investigate the spatial and temporal distributions of multiple -phytohormones simultaneously from one plant tissue sample. In this study, we develop a high-sensitivity and high-throughput method for the simultaneous quantitative analysis of 44 phytohormone compounds, covering currently known 10 major classes of phytohormones (strigolactones, brassinosteroids, gibberellins, auxin, abscisic acid, jasmonic acid, salicylic acid, cytokinins, ethylene, and polypeptide hormones [e.g., phytosulfokine]) from only 100 mg of plant sample. These compounds were grouped and purified separately with a tailored solid-phase extraction procedure based on their physicochemical properties and then analyzed by LC-MS/MS. The recoveries of our method ranged from 49.6% to 99.9% and the matrix effects from 61.8% to 102.5%, indicating that the overall sample pretreatment design resulted in good purification. The limits of quantitation (LOQs) of our method ranged from 0.06 to 1.29 pg/100 mg fresh weight and its precision was less than 13.4%, indicating high sensitivity and good reproducibility of the method. Tests of our method in different plant matrices demonstrated its wide applicability. Collectively, these advantages will make our method helpful in clarifying the crosstalk networks of phytohormones.

An Arabidopsis Secondary Metabolite Directly Targets Expression of the Bacterial Type III Secretion System to Inhibit Bacterial Virulence.

Plants deploy a variety of secondary metabolites to fend off pathogen attack. Although defense compounds are generally considered toxic to microbes, the exact mechanisms are often unknown. Here, we show that the Arabidopsis defense compound sulforaphane (SFN) functions primarily by inhibiting Pseudomonas syringae type III secretion system (TTSS) genes, which are essential for pathogenesis. Plants lacking the aliphatic glucosinolate pathway, which do not accumulate SFN, were unable to attenuate TTSS gene expression and exhibited increased susceptibility to P. syringae strains that cannot detoxify SFN. Chemoproteomics analyses showed that SFN covalently modified the cysteine at position 209 of HrpS, a key transcription factor controlling TTSS gene expression. Site-directed mutagenesis and functional analyses further confirmed that Cys209 was responsible for bacterial sensitivity to SFN in vitro and sensitivity to plant defenses conferred by the aliphatic glucosinolate pathway. Collectively, these results illustrate a previously unknown mechanism by which plants disarm a pathogenic bacterium.